Chemical reaction device, chemical reaction system, and chemical reaction method

ABSTRACT

A device is provided for a chemical reaction between molecules immobilized on a solid phase and molecules in a solution. A chemical analysis device is also provided to capture molecules in the solution and subsequently measure the captured molecules. Reaction efficiency and sample throughput are thereby improved. The chemical reaction and chemical analysis devices use a microfluidic device channel as a reaction vessel. The channel is provided with a particular molecule immobilized on an interior surface of the channel with an obstacle positioned against the flow. In a typical reaction vessel having an enzyme immobilized in the capillary&#39;s interior surface and glass beads as obstacles, a reaction solution can move either in one direction or back and forth to react with the immobilized enzyme. The flow of the reaction solution is not laminar such that a reaction between the particular molecule and the reaction solution proceeds at high efficiency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. application Ser.No. 10/854,018 filed on May 26, 2004 now U.S. Pat. No. 7,666,662, whichis related to U.S. application Ser. No. 10/750,886 filed on Jan. 5, 2004now abandoned, U.S. application Ser. No. 10/788,440 filed on Mar. 1,2004 now U.S. Pat. No. 7,445,755, and U.S. application Ser. No.10/790,063 filed on Mar. 2, 2004 now abandoned, the disclosures of whichare hereby incorporated by reference. The present application claimspriority from U.S. application Ser. No. 10/854,018 filed on May 26,2004, which claims priority from Japanese application JP 2003-196178filed on Jul. 14, 2003, the content of which is hereby incorporated byreference into this application.

FIELD OF THE INVENTION

This invention relates a chemical reaction device, a chemical reactionsystem, and a chemical reaction method. More specifically, thisinvention relates to a chemical reaction device, a chemical reactionsystem, and a chemical reaction method wherein a chemical reaction in aminute space is utilized. This invention also relates to a chemicalreaction device, a chemical reaction system, and a chemical reactionmethod wherein a biological molecule such as a nucleic acid or a proteinis involved in the reaction.

BACKGROUND OF THE INVENTION

Recent years have seen significant advances in the technology ofconstituting a minute space for conducting a chemical reaction in such aspace. One method of constituting such a minute space is use ofphotolithography which is a technology most frequently used in theproduction of a semiconductor. A microfluidic device can be constitutedby forming a groove in a silicon substrate by means of photolithographicprocess, and adhering another substrate over the groove to thereby forma minute channel for receiving a fluid which is actuated by the device.Various proposals have been made for use of such microfluidic device inchemical production (for example, Analytical Chemistry, 74, 3112-3117(2002)) and chemical analysis (for example, Analytical Chemistry, 73,2112-2116 (2001)).

Chemical reactions utilizing an enzyme immobilized on microparticleshave been investigated for various enzymatic reactions, and typicalprocesses include the process wherein microparticles having an enzymeimmobilized thereon are suspended in the solution for chemical reaction,and the process wherein such microparticles are used by filling in acolumn having a diameter much larger than the microparticles. Also knownin the art as a process utilizing a chemical reaction that takes placeat the interface between the microparticle and the liquid is thepurification of a particular molecule by using microparticles whereinmicroparticles having immobilized protein A or the like are filled in acolumn for use as an antibody purification column.

In the meanwhile, biological molecules are typically measured byimmobilizing a probe molecule in each well of a micro-well plate, anddispensing the sample in each well to measure the biological moleculecaptured by the probe molecule. In the case of ELISA, for example, anantibody is used for the probe molecule to quantitatively measure thebiological molecule in the sample. In this case, an antibody moleculewhich has affinity for the biological molecule is immobilized on theplate, and after capturing the biological molecule by the antibodymolecule, a high sensitivity measurement by enzymatic chemiluminescenceis conducted using an enzyme-labeled secondary antibody having affinityfor the biological molecule.

Another method for measuring a biological molecule that is indevelopment is a method wherein a plurality of probe molecules areimmobilized in different areas of the solid phase to measure thebiological molecule captured by each probe molecule. An example is theDNA chip wherein a plurality of probe DNAs are immobilized on a planarsubstrate of glass or the like for detection of nucleic acid molecules.In the case of this chip, a solution containing a fluorescence-labelednucleic acid molecule is placed on the chip for hybridization, and theamount of analyte nucleic acid molecule in the solution is determined bydetecting fluorescence on the chip. The methods often used for producingsuch a DNA chip include spotting of the DNA probe on a slide glass (forexample, Science, 270, 467-470 (1995)), and use of a photolithographicprocess and the sequential synthesis of the DNA by a photochemicalreaction (for example, Science, 251, 767-773 (1991)). Also proposed is aprotein chip wherein a plurality of proteins are simultaneously measured(for example, Analytical Biochemistry, 278, 123-131 (2000)). Devicesusing not the planar surface but a channel formed in the device havealso been proposed, and exemplary such devices include the one wherein aDNA probe has been immobilized in the capillary (for example, JapanesePatent Application Laid-Open No. 11-75812), and a probe array whereinprobe-conjugated microparticles have been arranged in the interior ofthe capillary (for example, Japanese Patent Application Laid-Open No.11-243997).

Processing a large amount of sample in a short time is generallydifficult in the chemical reaction system where molecules immobilized onthe solid phase are reacted with the molecules in the solution, or inthe chemical analysis system where molecules immobilized on the solidphase capture the molecules in the solution by chemically reacting withsuch molecules and the captured molecules are measured.

To be more specific, the methods mentioned in the Prior Art sectionincluding the method wherein microparticles are filled in a column, themethod using a micro-well plate, and the method using a DNA chip havebeen associated with the difficulty of establishing the sample flowwhile promoting reaction in the minute space, and as a consequence,improvement in the chemical reaction efficiency and reduction of theprocess time have been difficult.

SUMMARY OF THE INVENTION

In view of the problems to be obviated by the present invention asdescribed above, the present invention attempts to increase the reactionefficiency and reduce the reaction time in the chemical reaction wheremolecules immobilized on the solid phase are reacted with the moleculesin the solution, or in the chemical analysis where molecules immobilizedon the solid phase capture the molecules in the solution by chemicallyreacting with such molecules and the captured molecules are measured.Another object of the present invention is to improve the throughput tothereby increase the reaction efficiency when the sample is the one at alow concentration.

In the case of a minute space, an efficient and uniform chemicalreaction can be expected since the space where molecules involved in thechemical reaction can move by molecular diffusion is limited andprobability of the contact between the molecules is increased. Use of asmall space also enables reduction in the amount of the chemical reagentand the waste produced, as well as loss of the sample.

Accordingly, improvement of the reaction efficiency by using amicrofluidic device is contemplated wherein the micro-space in thechannel is used for the reaction space, and wherein a particularmolecule involved in the reaction is immobilized in the channel. When anarrow channel is used in such a case, the time of the solution passingover the area where the particular molecule has been immobilized will bereduced and diffusion of the reactant molecules in the solution will beinsufficient, presumably resulting in the difficulty of realizingsufficient reaction efficiency. On the other hand, a decrease in theflow rate to allow a sufficient time for the diffusion to therebyincrease the reaction efficiency would be associated with the risk ofreduced sample throughput.

In order to improve such a situation, the present invention provides achemical reaction device, a chemical reaction system, and a chemicalreaction method wherein a channel is used for the space of the chemicalreaction, and wherein a particular molecule is immobilized in itsinterior surface, and a structure or a flow obstacle is placed in thechannel. This structure or obstacle creates turbulence in the flow ofthe sample solution through the microfluidic device which is normally alaminar flow to remarkably increase the substantial diffusioncoefficient of the molecules in the solution, and as a consequence, adramatic increase in the reaction efficiency can be attained with nocompromise in the sample throughput. The time required for the reactionand analysis is also reduced, and handling of a sample at a lowconcentration is also enabled.

The chemical reaction device according to the present invention has acharacteristic feature that it has a channel for receiving the solutionand the structure accommodated in the channel, and a particular moleculewhich reacts with a substance included in a solution is immobilized inthe interior surface or lumen wall of the channel. The structure mayhave a diameter which is in the range of 30 to 90% of the diameter ofthe channel, and when volume allowed for the sample to flow in thechannel is V1, and interior volume of the channel is V2, the ratio V1/V2may be in the range of 0.4 to 0.95. The structure may be a fine particleor a strip, and the strip may be a wire or a rod. In the channel withinthe chemical reaction device, the flow of the solution may be at leastpartly a turbulent flow. The channel may be constituted from a grooveformed in a first substrate and a second substrate disposed to cover thegroove, or alternatively, the channel may be a lumen of a capillary.

The chemical reaction system according to the present inventioncomprises a thermal chamber for placing a reaction device foraccommodating a solution and a structure, and an introducer forintroducing the solution into the reaction device. The reaction devicehas a molecule immobilized on its interior surface to allow a chemicalreaction to take place between the immobilized molecule and thesubstance included in the solution introduced by the solutionintroducer. The chemical reaction system may further comprise a detectorfor detecting the reaction that takes place in the reaction device.

Furthermore, the chemical reaction method according to the presentinvention comprises the steps of: preparing a reaction device having aparticular molecule immobilized on its interior surface, and having astructure accommodated in its interior; introducing a solution into thereaction device; and allowing the particular molecule immobilized on theinterior surface to undergo a chemical reaction with a molecule includedin the solution; wherein, in the step of chemical reaction, the solutionmoves in relation to the structure. In this chemical reaction method,time required for the step of the chemical reaction may be at leastabout 10 minutes, and more specifically, this time may be designed atany time period in the range of at least about 5 minutes to at leastabout 15 minutes depending on the conditions desired for the chemicalreaction. A chemical reaction efficiency of considerable level isgenerally achieved when the time is designed to be at about 10 minutesor longer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C are schematic views showing the structures of thechemical reaction device and the chemical reaction system according anembodiment of the present invention;

FIGS. 2A and 2B are views showing the results of electrophoresisobtained by using the chemical reaction device and the chemical reactionsystem according an embodiment of the present invention;

FIGS. 3A and 3B are schematic views showing chemical reaction devicesfor comparison with the chemical reaction device according an embodimentof the present invention;

FIG. 4 is a graph showing an amount of the immobilized enzyme remainingin the chemical reaction device according to an embodiment of thepresent invention and the comparative chemical reaction device inrelation to elapsed time;

FIGS. 5A and 5B are schematic views showing the structures of thechemical analysis devices according an embodiment of the presentinvention;

FIGS. 6A and 6B are schematic views of the chemical analysis systemaccording to an embodiment of the present invention; and

FIG. 7 is a view showing the result of fluorometric analysis obtained byusing the chemical analysis device according an embodiment of thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Next, the present invention is described in further detail by referringto preferred embodiments of the present invention.

FIGS. 1A to 1C are views schematically showing the structure of thechemical reaction device and the chemical reaction system according tofirst embodiments of the present invention. FIG. 1A is a partialexploded view of an exemplary reaction vessel in the chemical reactiondevice according to an embodiment of the present invention. Thisreaction vessel comprises a channel defined in capillary 101 having anenzyme 102 immobilized on its interior surface and glass beads 103filled in its interior. The glass beads 103 function as structuresobstructing the flow through the interior or the lumen of the capillary101. Enzymatic reaction can be induced by passing the reaction solutioncontaining the molecule which is a substrate for the enzymatic reactionthrough the lumen of this capillary 101. This reaction vessel isapplicable to a wide variety of enzymatic reactions, and the reactionvessel described herein as an example is the one having trypsinimmobilized thereto used for the purpose of proteolysis. The proteolyticproduct produced by using this reaction vessel can be used, for example,for fingerprinting of the protein, or for identification of the unknownprotein by measuring the molecular weight with a mass spectrometer. Thecapillary 101 having the trypsin immobilized on its interior surface canbe produced, for example, by the procedure as described below. Thecapillary used in this embodiment is a fused quartz capillary having aninner diameter of 150 micron. First, the capillary is washed with purewater to clean the interior surface of the capillary to therebyintroduce hydroxy group, and 1N aqueous solution of sodium hydroxide at80° C. is passed through the capillary for 10 minutes, and the capillaryis again washed with pure water until the pH returns neutral. Next, 1%aqueous solution of 3-aminopropyltrimethoxysiane is introduced in thecapillary to introduce amino group on the interior surface of thecapillary, and the reaction is allowed to proceed at room temperaturefor 10 minutes. The capillary is then washed with pure water, andallowed to stand in an oven at 120° C. for 60 minutes. Next, 500 mMsuccinic anhydride (in 1-methyl-2-pyrrolidone) is introduced in thecapillary to introduce carboxyl group on the interior surface of thecapillary, and the reaction is allowed to proceed 50° C. for 60 minutes.The capillary is then thoroughly washed with pure water. Next, 20 mMN-hydrosuccineimide and 100 mMN-ethyl-N′-3-methylaminopropylcarbodiimide (in 0.1M borate buffer (pH6.2)) are introduced in the capillary to activate the carboxyl groupthat has been introduced on the capillary, and after leaving it at roomtemperature for 60 minutes, the capillary is washed with 0.1M boratebuffer (pH 6.2) and pure water. Finally, 50 mg/mL trypsin (in 0.1Mborate buffer (pH 6.2)) is introduced in the capillary to immobilizetrypsin, and after leaving it in the refrigerator at 4° C. for one day,the capillary is washed with 10 mM Tris-HCl buffer solution (pH 8.0).Until its use, the capillary is stored at 4° C. with 10 mM Tris-HClbuffer solution (pH 8.0) filled in its interior capillary. The capillaryis cut immediately before its use at an appropriate length, for example,at 20 cm, and the obstacle are filled in its interior. The obstacle usedin this embodiment is glass beads 103, and more specifically, the glassbeads which had passed through a 126 micron mesh sieve but which failedto pass through a 105 micron mesh, and which had been ultrasonicallywashed. The size of the glass beads 103 may be either consistent orinconsistent. In addition, the beads used for the obstacle may comprisea material other than glass such as a resin, and the beads may have anydesired configuration such as a sphere, an ellipsoid, or a polyhedron.When the size of the beads is defined by using the term “diameter”, thisterm refers to the diameter in the case of a sphere, the major axis inthe case of an ellipsoid, and in the case of a polyhedron, the longestof the line segment extending between a point on the surface of and thepoint symmetrically opposite to that point in relation to the center ofthe polyhedron.

Alternatively, the obstacle may comprise a stainless steel wire having adiameter of about 100 microns which has been cut. FIG. 1B shows anembodiment of the reaction vessel wherein a stainless steel wire hasbeen used for the obstacle. This reaction vessel comprises a channelwherein an enzyme 105 is immobilized on the inner surface of a capillary104, and a stainless steel wire 106 having a diameter of 100 microns isaccommodated in the interior of the capillary 104. When such wire orsimilar obstacle which is a strip member, cylindrical member, or thelike is used, the obstacle may be configured whether it is moved by theflow of the solution or by other mechanical means.

In the case of a microparticle-filled column wherein the column isfilled with the structure, and the structure comprises microparticleshaving a diameter of several microns which is by far smaller than thediameter of the capillary, the void where the substrate-containingsolution can flow will be extremely small, and the pressure loss will beextremely high. In such a case, the flow of the solution will be alaminar flow with no considerable increase in the substantial diffusioncoefficient, and the substrate will confront with the difficulty inreacting with the enzyme 102 immobilized on the interior surface, and asa consequence, the reaction efficiency will be reduced. For example,when a capillary having an inner diameter of 150 microns is used for thereaction vessel, the optimal diameter range of the beads used for theobstacle accommodated in the capillary is in the range of about 50micron to about 130 micron so that the beads will not be too smallcompared to the capillary while not too big to be received by thecapillary and will enable formation of the solution flow thataccelerates the chemical reaction. In other words, the beads may have adiameter which is at least about 30% and at most about 90% of thediameter of the capillary, and in this case, the flow of the reactionsolution formed in the capillary will not be a laminar flow but a flowin transition from the laminar flow to the turbulent flow or a turbulentflow. When the flow in the capillary is a laminar flow, a considerabletime is required for the substrate to move from the center of the flowto the vicinity of the channel wall, while all molecules will bepromoted to collide with the channel wall in the case of a turbulentflow or a flow in transition from the laminar flow to the turbulent flowincluding partial turbulent flow and the efficiency of the chemicalreaction is thereby improved. In this case, the plurality of beads inthe interior of the capillary may be arranged either such that thecenters of the beads are aligned on a straight line or such that thecenters of the beads are not aligned on a straight line as shown in FIG.1A.

When considered on the basis of the void, namely, the ratio of theinterior volume of the capillary or the channel excluding the volume ofthe structure (obstacle) to the entire interior volume of the capillaryor the channel, namely, the ratio represented by the formula: V1/V2 whenV1 is the volume of the space in the capillary where the sample can flowand V2 is the interior volume of the capillary if no structure(obstacle) were present, the void of the microparticle-filled columnwould be close to the void in the case of the hexagonal closest packingwhich is the closest packing in a free space with the void of 0.26. Asdescribed above, the flow of the solution is a laminar flow in the caseof the microparticle-filled column with a low substantial diffusioncoefficient, and the substrate has difficulty in reaching the enzyme 102immobilized on the interior surface. As a consequence, the reactionefficiency will be insufficient. In the present invention, the voidshould be larger than that of the microparticle-filled column in orderto form a flow of the solution that allows the chemical reaction, andthe adequate range for the void is about 0.5 to about 0.7 in the casewhen beads are used for the obstacle. When the obstacle is not in theform of spheres or ellipsoids but a continuous strip as shown in FIG.1B, design freedom of the void is significantly higher, and a void inthe range of about 0.4 to about 0.95 can be provided. When the obstacleallows provision of the void of the range as described above, the flowof the reaction solution created will not be a laminar flow but aturbulent flow or a flow in a transition state from the laminar flow tothe turbulent flow including partial turbulence, and the efficiency ofthe chemical reaction can be improved as in the case of using the beadsof predetermined diameter.

FIG. 1C is a view schematically showing the structure of the entirechemical reaction system produced by using the chemical reaction deviceaccording to the first embodiment of the present invention. A reactionvessel 110 is the one described by referring to FIG. 1A comprising thecapillary 101 having the enzyme 102 immobilized on its interior surfaceand filled with the glass beads 103. On opposite ends of the reactionvessel 110 are provided connectors 111 and 111′ which prevent exit ofthe glass beads 103 filled in the reaction vessel 110 from the reactionvessel 110 while feeding the solution to the reaction vessel 110, andthese connectors 111 and 111′ also join the reaction vessel 110 withfeed capillaries 112 and 112′. A sample tube 113 filled with thereaction solution containing the molecule which serves the substrate forthe enzymatic reaction is provided at the end of the feed capillary 112,and a syringe 114 of a syringe pump 115 is provided at the end of thefeed capillary 112′. When the syringe pump 115 is operated, the reactionsolution in the sample tube 113 is introduced in the reaction vessel110, and the reaction solution introduced subsequently moves either inone direction or back and forth in two directions corresponding to theoperation of the syringe pump 115. Such operation can promote thereaction between the enzyme immobilized on the interior surface of thereaction vessel 110 and the substrate in the reaction solution. Theentire reaction system may be placed in a thermal chamber 116 in orderto maintain the temperature at a constant level during the chemicalreaction. When the syringe 114 and the syringe pump 115 are notresistant to temperature load, the syringe 114 and the syringe pump 115may be placed outside the thermal chamber 116. In the case of theproteolytic reaction using the trypsin-immobilized glass beads asdescribed by referring to FIG. 1A, the sample reaction solution of about1 μL to about 300 μL can be handled with no inconvenience. For example,when the syringe pump 115 is operated at the volume flow rate of about 1to about 100 μL per minute, behavior of the reaction solution in thereaction vessel 110 will be a flow in transition from the laminar flowto the turbulent flow and not a laminar flow, with an increasedsubstantial diffusion constant of the substrate molecule.

Next, the use of the system of the present invention is described.Cytochrome C, which is a protein, was decomposed in this chemicalreaction system by trypsin. The reaction solution was 10 μL of 0.2 mg/mLcytochrome C (in 10 mM Tris-HCl buffer solution (pH 8.0)), and thissolution was moved back and forth in the reaction vessel at a flow rateof 10 μL per minute and at a reaction temperature of 37° C. for theproteolysis by trypsin. The reaction time was 0 minute (no reaction), 15minutes, 30 minutes, or 60 minutes. To 5 μL of the reaction solutioncollected after the reaction was added 2 μL of loading buffer (313 mMTris-HCl, 10% SDS, 10% mercaptoethanol, 30% glycerol, 0.01% bromophenolblue, pH 6.8), and the mixture was heated to 95° C. for about 2 minutes.Of such mixture, 3 μL was used as a sample in the electrophoresis. Theelectrophoresis was conducted by using 15% polyacrylamide gel havingsodium dodecyl phosphate (SDS) added thereto, and an electrophoreticbuffer (25 mM Tris-HCl, 192 mM glycine, 0.1% SDS, pH 8.5). The currentin the electrophoresis was set at 20 mA, and after two hours ofelectrophoresis, the gel was stained with Coomassie Brilliant Blue for 1hour, and destained with 10% acetic acid-40% methanol solution for 2hours to obtain the electropherogram. For a comparison purpose, 1 μL oftrypsin solution at a concentration 1 mg/mL and 39 μL of pure water wereadded to 50 μL of 2 mg/mL cytochrome C (10 mM Tris-HCl solution (pH8.5)), and the mixed solution was left in a reaction tube with nomovement at 37° C. to promote the proteolytic reaction. The reactiontime use was 0 hour (no reaction), 1 hour, 4 hours, 16 hours, and 20hours. The reaction products were electrophoresed as in the case of theproducts obtained by using the chemical reaction system to also obtainthe electropherogram. FIG. 2 shows the results of the electrophoresiswhen cytochrome C was decomposed with trypsin as described above. FIG.2A shows the electropherograms obtained by using the chemical reactionsystem of FIG. 1, and FIG. 2B shows the results obtained by leaving thereaction solution in the reaction tube with no movement. When thechemical reaction system shown in FIG. 1 is used, the band fromcytochrome C observed for the reaction time of 0 minute (no reaction) isnot observed in the samples of reaction times 15 minutes, 30 minutes,and 60 minutes as shown in FIG. 2A, and this indicates that thecytochrome C has been decomposed by trypsin. While no band for theproteolytic product are observed, such absence is estimated to be due tothe increased variety of fragments each comprising a reduced amount aswell as due to the short length of such products. In contrast, when thereaction is promoted in the reaction tube with no solution movement, theresults are substantially similar whether the reaction times is 1 hour(60 minutes) or 0 hour (no reaction) as shown in FIG. 2B. While thedensity of the band decreases with the increase in the reaction timeindicating the progress of the decomposition by trypsin, the band doesnot completely disappear even after the reaction time of as long as 20hours indicating the presence of the cytochrome C that had not beendecomposed. These results indicate that proteolytic reaction of thecytochrome C by trypsin is fully completed in the case of the reactionvessel of the present invention in 15 minutes. Decomposition of theprotein in a solution by trypsin generally takes one day for completionwhen the solution is not moved. In contrast, the reaction conducted bythe method of the present invention is completed in about 10 minutes toabout 20 minutes, realizing a dramatic reduction of the time requiredfor the analysis. It is to be noted that while the chemical analysissystem has been described in the foregoing for the embodiment whereinthe chemical reaction conducted is an enzymatic reaction using an enzymeas the probe immobilized in the interior of the capillary, the reactionmay also be the one wherein a nucleic acid is used for the probe.Exemplary molecules which may be immobilized in the reaction vesselinclude biological molecules other than such enzyme and nucleic acid,and exemplary analytes include nucleic acids, proteins, and otherbiological molecules. When the chemical reaction carried out is anenzymatic reaction, the reaction that had undergone the enzymaticreaction is, for example, recovered from the capillary. When thechemical reaction carried out is the binding to the probe of thesubstance which is specific to the probe, the specific substance thatbecame bonded to the probe is collected or detected after the reaction.

Compared to the case wherein the molecule involved in the chemicalreaction is immobilized on the glass beads or other obstacles, thepresent invention wherein the molecule is immobilized on the interiorsurface of the reaction vessel is more tolerant to the operation thatmay cause damages to the molecule-immobilized surface such asintroduction of the molecule-immobilized glass beads into the reactionvessel, and ease of the handling of the beads is thereby realized. Thisenables production of the reaction system at a reduced cost and atimproved reproducibility. Another problem is the peeling or separationof the immobilized molecule induced by the flow of the reaction solutionat the surface of the obstacle such as glass beads having a curvedsurface, and as a matter of fact, the molecule immobilized on a curvedsurface receive a force larger than the molecule immobilized on thenon-curved surface like the interior surface of the channel (orcapillary), and the molecule involved in the reaction that isimmobilized on the surface is relatively easily peeled off the surfacein the case of a curved surface. This is always a serious problem whenthe molecule is immobilized by using the physical adsorption, namely, byutilizing the adsorption caused by intermolecular force. Whileimmobilization of the molecule using physical adsorption is quite usefuldue to the simple procedure, no covalent bond is present between thesolid phase and the immobilized molecule in the case of the physicaladsorption, and accordingly, the molecule immobilized by the physicaladsorption cannot endure the physical load applied to the molecule bythe fluid and the like and the molecule is liable to become peeled offthe surface. However, in the case of the present invention, themolecules are immobilized in a manner resistant to the peeling in thereaction process, and a high reaction efficiency is thereby realized.

An experiment was conducted to evaluate the liability of the immobilizedmolecule to become peeled off the surface when the molecule isimmobilized by physical adsorption. The system used was the one the sameas the one shown in FIG. 1C except that the enzyme was immobilized onthe interior surface of the capillary. Shown in FIG. 3 are schematicviews of the chemical reaction devices wherein the molecule has beenimmobilized by physical adsorption. FIG. 3A is a partial exploded viewof the channel of the chemical reaction devices wherein an enzyme hasbeen immobilized by physical adsorption on the interior surface of thecapillary. An enzyme 202 is immobilized on the interior surface of thecapillary 201 by physical adsorption, and glass beads 203 areaccommodated in the interior of the capillary 201 as an obstacle for theflow. FIG. 3B is a partial exploded view of the channel of the chemicalreaction devices wherein the enzyme has been immobilized not on theinterior surface of the capillary but on the glass beads by physicaladsorption. In this experiment, the chemical reaction devices areproduced by immobilizing trypsin as an exemplary enzyme. Theimmobilization of trypsin on the surface of the solid phase is carriedout, for example, by the procedure as described below. In the case whentrypsin is immobilized on the interior surface of the capillary 201 asshown in FIG. 3A, the capillary is first washed with pure water, and the1N aqueous solution of sodium hydroxide at 80° C. is passed through thecapillary for 10 minutes, and the capillary is again washed with purewater until the pH returns to neutral. A hydroxyl group is introduced onthe interior surface of the fused quartz capillary by the treatment withthe sodium hydroxide, and the surface assumes negative charge. Next, 1mg/mL solution of trypsin in 0.05M Tris buffer solution (pH 7.0) ispassed through the capillary for 1 hour at room temperature, and thecapillary is washed with 0.05M Tris buffer solution (pH 7.0). Sincetrypsin has a calculated isoelectric point of about 8.5, it assumes apositive charge in the solution of pH 7.0, and as a consequence, itbecomes immobilized on the glass surface by physical adsorption. Thethus produced capillary may be stored until its use by filling 0.05MTris buffer solution (pH 7.0) in the interior of the capillary andmaintaining at 4° C. The chemical reaction device of FIG. 3A can bereadily produced immediately before its use by cutting the capillary atan appropriate length and the accommodating the glass beads in theinterior. The glass beads having the enzyme immobilized by physicaladsorption may also be produced by using the similar reaction conditionsto thereby immobilize the enzyme on the surface of the glass beads. Thereaction vessel schematically shown in FIG. 3B can be produced byfilling the glass beads 205 in the capillary 204. Each of the thusproduced two reaction vessels is placed as a component in the chemicalreaction system of FIG. 1B produced by using the same components as thefirst embodiment as described above except for the capillary and theglass beads, and protein-free Tris buffer solution (pH 7.0) is passedthrough the reaction vessel to thereby measure the amount of the trypsinremaining in the reaction vessel after the passage. The buffer solutionis used at an amount of 100 μL, and the buffer solution is passed backand forth through the reaction vessel at a flow rate of 50 μL perminute. Amount of the trypsin that was peeled off into the buffersolution is measured by varying the time of the buffer reciprocal cycle.In view of the difficulty of the quantitative protein evaluation at aminute amount, trypsin labeled with a radioisotope C¹³ was used in thisevaluation. First, amount of trypsin that had been immobilized isdetermined by measuring the radiation from the trypsin solution used forthe immobilization before and after the trypsin immobilization, andcalculating the difference in the amount of the trypsin. The radiationof the buffer solution that had passed through the capillary is thenmeasured to determine the amount of trypsin in the buffer solution thathad been incorporated by the peeling of the trypsin. This amount wascompared with the amount of the previously calculated immobilizedtrypsin.

FIG. 4 is a graph showing the time course of the amount of theimmobilized trypsin remaining in the chemical reaction device, and thevalue has been normalized by taking the amount of trypsin that had beeninitially immobilized in each chemical reaction device as “1”. The curveindicated with blank circle 210 represents the data of the chemicalreaction device of FIG. 3A comprising the capillary 201 having theenzyme 202 being immobilized on its interior surface and the glass beads203 filled therein, and the curve indicated with blank triangle 211represents the data of the chemical reaction device of FIG. 3Bcomprising the capillary 204 filled with the enzyme-immobilized glassbeads 205. For example, when the reaction time is 20 minutes, about 80%and about 50% of the trypsin are remaining in each chemical reactiondevice, and this indicates that a higher reaction efficiency is realizedin the chemical reaction device having the enzyme 202 immobilized on theinterior surface of the capillary 201 with the greater amount ofeffective trypsin. Although the data has been presented for the devicewherein the molecule has been immobilized by utilizing physicaladsorption, the situation is similar for the case when the molecule isimmobilized by means of covalent bond, and higher reaction efficiency isrealized when the enzyme is immobilized on the interior surface of thecapillary. It is also to be noted that, when the results shown in FIGS.2 and 4 are taken into consideration, the time of the chemical reactionshould be set at any time between at least about 5 minutes and at leastabout 15 minutes, since the chemical reaction should proceed to thesufficient level, and simultaneously, the starting of the reactionefficiency loss by the peeling of the immobilized molecule should beconsidered. A chemical reaction efficiency of predetermined level isgenerally realized when a time of about 10 minutes is allowed for thechemical reaction.

Furthermore, when the enzyme is immobilized on the microparticles andthe microparticles are suspended in a solution containing the substratemolecule for the chemical reaction, full suspension of themicroparticles in the solution is generally difficult. In other words,it is difficult to bring the microparticles in full contact with thesubstrate molecule included in the solution to induce the chemicalreaction. In addition, in use of a column filled with enzyme-immobilizedmicroparticles, the microparticles are filled at a high rate in thereaction vessel with insufficient void as described above, and thereaction solution can only flow at a slow speed and the flow will be alaminar flow with reduced substantial diffusion speed. As a consequence,an increase in the reaction efficiency is difficult, and a high pressurepump will be required if the flow rate were to be increased. Incontrast, the present invention comprises a device wherein a particularsubstance is immobilized on the interior surface of the reaction vesselso that the molecule reacting with the particular substance is forced topass through a narrow space, and at the same time, wherein the flowformed is not a laminar flow and turbulent flow is at least partlyinduced, thereby enabling a highly efficient reaction to take place in ashort time. Furthermore, since the reaction vessel of the presentinvention is constituted such that the reaction vessel has theparticular substance immobilized on its interior surface and a structureor an obstacle accommodated in the interior as described above, thereaction vessel also enjoys the merit of reduced peeling or separationof the immobilized molecule from the surface to which it has beenimmobilized, which has been a serious problem in the system where themolecule is immobilized on the microparticles.

FIG. 5 schematically shows the structure of the chemical analysis deviceaccording to the second embodiment of the present invention. FIG. 5A isa total view of DNA measurement device in the form of a chip. Thisdevice comprises a flat slide glass 302 and a polydimethylsiloxane(PDMS) substrate 301 adhered to the slide glass 302, and the PDMSsubstrate 301 has a channel 303 formed therein to provide the site ofthe chemical reaction and simultaneously, the site of the detection. Theslide glass 302 has a size of 25 mm×75 mm, and a thickness of about 1mm. A nonfluorescent product comprising crown glass was used for theslide glass 302 for the subsequent fluorometric measurement. The PDMSsubstrate 301 has a thickness of about 2 mm, and it covers the slideglass 302 except for the margin of about 2 mm along the long sides ofthe slide glass 302. The regions of the slide glass 302 not covered bythe PDMS substrate 301 are used as the gripper region for holding thedevice while it is inserted in the DNA chip scanner in the subsequentfluorometric measurement. The channel 303 has a cross section with thesize of 150 micron×150 micron and the length of 4 cm. The channel 303 isformed at one end with a solution inlet 304 for introducing an analytesample solution and washing solution, and at the other end with aconnection port 305 used for connection with an outer pump which is usedto introduce the solution and to pass the solution back and forth in thechannel. This device is produced by the procedure described, forexample, in Electrophoresis, 22, 328-33 (2001) by pouring unreacted PDMSin the mold produced by photolithographic means having the shape of theresulting PMDS substrate for curing, releasing the cured PDMS substrate301 from the mold, and adhering the PDMS substrate 301 with the slideglass 302. FIG. 5B is an exploded view of a part of the channel 303which functions as the chemical reaction site as well as the measurementsite of shown in FIG. 5A. The channel 303 is defined between the PDMSsubstrate 301 and the slide glass 302. PDMS substrate 301 hasprojections 311 which protrude into the channel 303 from the upper sideof the channel 303. This projection 311 has the effect of disturbing theflow of the reaction solution to increase the substantial diffusioncoefficient of the substrate. Beside the fact that an excessively thinstructure is difficult to provide on the PDMS substrate 301, thestructure should have a substantial size to create turbulence in theflow of the solution passing through the channel 303 to thereby increasethe reaction speed. An adequate size contemplated for the projection isat least about ⅓ of the width or the height of the channel. In theexample shown in FIG. 3B, the projection 311 is a cube with the lengthof sides of 75 microns. The projections 311 are disposed at an intervalof 200 microns. In this example, a DNA probe 310 is spotted on the sideof the slide glass 302 of the channel 303. While the spots are notlimited for their size, the spot typically has a diameter of about 50microns to about 100 microns. The spotting of the DNA probe 310 may beconducted by using a commercially available spotter. The spotting ofthis DNA probe on the slide glass 302 may be completed before theadhesion of the PDMS substrate 301 to the slide glass 302. DNA probes310 are provided at an interval of 200 micron as in the case of theprojections 311.

FIG. 6 is a view schematically showing the constitution of the chemicalanalysis system wherein the DNA measurement device of FIG. 5 has beenincorporated. The DNA measurement device 321 having the solution inlet304 for introducing the analyte sample solution and the washingsolution, the channel 303, and the connection port 305 is accommodatedin the thermal chamber 322. To the connection port 305 is connected to aconnection capillary 329 by the intervening connector 323. Theconnection capillaries 329, 330, and 331 are divided by a three wayvalve 327 into three sections. A capillary section 331 is connected atone end with a syringe 325 on a syringe pump 326, and a capillarysection 330 is at one end connected to waste reservoir 328. In analyzingDNA, the analyte fluorescence-labeled target DNA is captured by a DNAprobe by means of hybridization which is a type of chemical reaction,and the captured fluorescence-labeled target DNA is measured byfluorescence, as in the case of the normal DNA chip. This fluorometricmeasurement may be accomplished by various methods such as those using afluorescence microscope. An embodiment using a system like a DNA chipscanner is shown in FIG. 6B. The beam exiting from a laser 351 isreflected at a dichroic mirror 355, passes through a dichroic mirror353, and condensed by a lens 354 to be directed to the DNA measurementdevice. The fluorescence produced by fluorescence-labeled target DNAupon irradiation of the fluorescence-labeled target DNA with the laserbeam is condensed by the lens 354, reflected by the dichroic mirror 353,passes through an optical filter 356, and measured by a photomultiplier352 for analysis by a personal computer 357. In order to conduct themeasurement for each DNA probe in the DNA measurement device, laser beamscanning may be carried out by moving the DNA measurement, or byconstituting the scanner-like system as described above so that apart ofthe system is movable and moving this movable part of the system. It isto be noted that the scanner-like system similar to this system can beused in the embodiments shown in FIGS. 1A, 1B, and 1C. To be morespecific, in the embodiments shown in FIGS. 1A, 1B, and 1C, when thechemical reaction is the binding between a probe and a substance whichspecifically reacts with the probe, the fluorescence emitted by thefluorescence label which is directly or indirectly bonded to the probecan be detected by the system like the one shown in FIG. 6B afterremoving the structure such as beads or the wire accommodated in thecapillary after the completion of the reaction.

An embodiment of DNA analysis using the DNA measurement device 321 wasconducted. The DNA probes used were synthetic DNAs having a length of 18bases which is a part of the nucleotide sequence on the antisense sideof the exons of p53 (Exons 1 and 3 were not used; 10 types in total;designated probes 1 to 10). The target DNAs used were synthetic DNAshaving a length of 18 bases which are fully complementary to the DNAprobes, and which are labeled with Cy3 fluorescence marker (10 types intotal, target 1 to 10). The melting temperature of the complementary DNAprobe and the target DNA was about 70° C. The sequences are as describedbelow.

Sequence of DNA probe 1: 5′-TGTCACCGTCGTGGAAAG-3′ (SEQ ID NO: 1)Sequence of DNA probe 2: 5′-ATCTGACTGCGGCTCCTC-3′ (SEQ ID NO: 2)Sequence of DNA probe 3: 5′-AAGAAGCCCAGACGGAAA-3′ (SEQ ID NO: 3)Sequence of DNA probe 4: 5′-GCCTCACAACCTCCGTCA-3′ (SEQ ID NO: 4)Sequence of DNA probe 5: 5′-TCATAGGGCACCACCACA-3′ (SEQ ID NO: 5)Sequence of DNA probe 6: 5′-ATGATGGTGAGGATGGGC-3′ (SEQ ID NO: 6)Sequence of DNA probe 7: 5′-CCCTTTCTTGCGGAGCTT-3′ (SEQ ID NO: 7)Sequence of DNA probe 8: 5′-TTTCTTCTTTGGCTGGGG-3′ (SEQ ID NO: 8)Sequence of DNA probe 9: 5′-CCTGGGCATCCTTGAGTT-3′ (SEQ ID NO: 9)Sequence of DNA probe 10: 5′-ATGGCGGGAGGTAGACTG-3′ (SEQ ID NO: 10)Sequence of DNA target 1: 5′-CTTTCCACGACGGTGACA-3′ (SEQ ID NO: 11)Sequence of DNA target 2: 5′-GAGGAGCCGCAGTCAGAT-3′ (SEQ ID NO: 12)Sequence of DNA target 3: 5′-TTTCCGTCTGGGCTTCTT-3′ (SEQ ID NO: 13)Sequence of DNA target 4: 5′-TGACGGAGGTTGTGAGGC-3′ (SEQ ID NO: 14)Sequence of DNA target 5: 5′-TGTGGTGGTGCCCTATGA-3′ (SEQ ID NO: 15)Sequence of DNA target 6: 5′-GCCCATCCTCACCATCAT-3′ (SEQ ID NO: 16)Sequence of DNA target 7: 5′-AATCTCCGCAAGAAAGGG-3′ (SEQ ID NO: 17)Sequence of DNA target 8: 5′-CCCCAGCCAAAGAAGAAA-3′ (SEQ ID NO: 18)Sequence of DNA target 9: 5′-AACTCAAGGATGCCCAGG-3′ (SEQ ID NO: 19)Sequence of DNA target 10: 5′-CAGTCTACCTCCCGCCAT-3′ (SEQ ID NO: 20)

Immobilization of the DNA probe can be accomplished by various methods.In this embodiment, the DNA probe was immobilized by using a spotter,and the DNA probe solution was spotted on a slide glass coated withpoly-L-lysine by the tip of a spotter. The DNA probes were spotted in aline from the side of the solution inlet 304 to the side of theconnection port 305 in the order of from DNA probe 1 to DNA probe 10.After the spotting, the slide glass was exposed to steam for severalseconds for rehydration, and the DNA probe was immobilized byirradiating with UV of 60 mJ in a UV cross linker. In order to preventnon-specific hybridization, the slide glass was immersed in a blockingsolution for 20 minutes, and washed with pure water and ethanol in thisorder. The blocking solution was prepared by mixing 335 mL of1-methyl-2-pyrrolidone, 5.5 g of succinic anhydride, and 15 mL of 1Msodium borate (pH 8.0). The hybridization buffer solution used was4×SSC−0.1% SDS solution, and the solution was prepared so thatconcentration of the target DNA was 1×10⁻¹⁰M. Immediately before theanalysis, the sample solution containing the target DNA was heated to94° C. for 2 minutes for denaturation, and the solution was cooled onice. The analysis was conducted by the procedure as described below.First, the thermal chamber 322 was set at 45° C., and left at thistemperature for 10 minutes for stabilization. Next, 10 μL of the samplesolution that had been kept on ice was introduced into the solutioninlet 304. The three way valve 327 was opened in the direction tocommunicate the syringe 325 and the connection port 305, and the syringepump 326 was operated to introduce and pass the sample solution to andthrough the channel 303 having the DNA probes spotted therein. Since thechannel 303 has an interior volume of up to 1 μL, the sample solutionthat had been introduced in the channel 303 passes through theconnection port 305 to become introduced in the capillary 329. Thesyringe pump 326 was stopped when the syringe 325 has moved 10 μL whichis the volume identical with the volume of the sample. The capillary 329between the three way valve 327 and the connection port 305 was designedto have an interior volume exceeding 10 μL so that the sample solutionwould not reach the three way valve 327. The piston of the syringe 325was moved back and forth by the syringe pump 326 to move the samplesolution back and forth in the channel 303 having the DNA probeimmobilized therein to thereby promote the hybridization. In thisembodiment, the volumetric flow rate was 10 μL per minute. It isimportant that the volumetric flow rate used is determined so that aturbulent flow is at least partly induced in the channel 303 by theprojection (the projection 311 described for FIG. 5), and thehybridization is accelerated by a turbulent flow which assists reachingof the target DNA to the DNA probe. After reacting for 10 minutes, thesample solution is pulled to the side of the syringe 325 past the threeway valve 327, and the three way valve 327 was turned to connect thesyringe 325 and the waste reservoir 328 to discard the sample solutionthat had undergone the reaction in the waste reservoir 328. Next, thesystem was washed with 30 μL of 1×SSC−0.03% SDS solution, 30 μL of0.2×SSC, 30 μL of 0.05×SSC, and 30 μL, of pure water. The washing wasconducted by supplying the washing solution in a procedure similar tothe washing except that the volumetric flow rate of the washing solutionwas increased to 30 μL per minute, washing time was 1 minute, and thesolution was subsequently discarded in the waste reservoir 328. Thetemperature during the washing was 45° C. which is lower than themelting temperature, and most of the target DNA that had been capturedon the DNA probe by the specific hybridization endured the peeling andremained on the surface. After the washing, the DNA measurement device321 was collected from the thermal chamber 322, and fluorescence wasmeasured by a DNA chip scanner. The slide glass and the PDMS resin whichhad been used for producing the DNA measurement device was substantiallytransparent to visible light and did not emit the fluorescence, andtherefore, the target Cy3 fluorescence was not interfered. FIG. 7schematically shows the results of the scanning of the DNA measurementdevice 321 after the reaction with the DNA chip scanner in an explodedview of the part of the channel 303. The results shown in FIG. 7 is theanalysis of the sample solution which only contain target 5.Fluorescence was observed only for spot where probe 5 complementary totarget 5 had been spotted while no fluorescence was observed for otherspots (indicated by white dotted line) of the probe other than the probe5, indicating that the results observed reflected hybridization thatdepended on sequences. As described above, hybridization reaction whichis generally a time-consuming step could be accomplished in about 10minutes by providing a projection as the structure near the DNA probe inthe channel and inducing a turbulent flow in the flow of the sample.

In this embodiment, a combination of PDMS resin and a slide glass forthe material was used in producing the device. The device can be alsoproduced by adhering a glass component with another glass component, orby using other resins (for example, polymethyl methacrylate). Inaddition, surface irregularities of various configurations in thechannel can be utilized in addition to the simple repetition of cubicprojections. Since the turbulent flow occurs in the vicinity of thestructure, the distance between the site of spotting the DNA probe andthe structure is critical. Since the distance required for the formationof the established flow in the case of a simple channel is believed tobe a distance about several folds of the width of the channel, thedistance between the center of the spot of the DNA probe and the centerof the structure in the direction of the solution flow is preferablydesigned to be up to about threefold the width of the channel in theplane substantially perpendicular to the direction of the solution flow,namely, the height or distance between the interior surface of thechannel where the spot has been provided and the opposite interiorsurface of the channel. Alternatively, glass beads and other obstaclesmay be accommodated in the channel having the DNA probe immobilizedtherein instead of providing a structure in the channel as in the caseof the first embodiment to thereby cause a turbulent flow in the flow ofthe solution to thereby increase the hybridization speed. Thefluorescence can also be directly measured in this case with an opticalmeans such as a DNA chip scanner if the obstacles are opticallysubstantially transparent, and if not substantially transparent, theobstacles may be removed after the reaction and before the measurement.While the DNA measurement device described in this embodiment has been achemical analysis system wherein a DNA is used for the prove to measurethe DNA, an immunoanalysis utilizing an antibody or like may also becarried out, and the molecule immobilized may comprise proteins otherthan the nucleic acid or the antibody. The analyte may also comprise anucleic acid, a protein, and a biological molecule. While the moleculemeasured in this embodiment is the captured molecule, a chemicalanalysis device utilizing an enzyme sensor, for example, is alsocontemplated wherein the product of the specific reaction between theimmobilized enzyme and the analyte molecule is detected in anotherregion in the downstream. While the captured molecule is detected byusing fluorometry, a highly sensitive chemical analysis system can alsobe constituted by using measurements such as chemiluminescence, colorreaction, and surface plasmon scattering.

The present invention has realized a chemical reaction device, achemical reaction system, and a chemical reaction method which exhibithigh reaction efficiency as well as high sample throughput. The presentinvention has also realized a chemical reaction device, a chemicalreaction system, and a chemical reaction method which are useful whenthe reaction should be completed in a short time and the number of thetarget molecule is small. The present invention has also realized achemical reaction device, a chemical reaction system, and a chemicalreaction method which are highly sensitive.

1. A chemical reaction system, comprising: a thermal chamber for placinga reaction device for accommodating a solution and a structure, and anintroducer for introducing said solution into said reaction device,wherein said reaction device has at least a particular moleculeimmobilized on its interior surface to allow a chemical reaction to takeplace between said immobilized molecule and a substance included in saidsolution introduced by said solution introducer, wherein the reactiondevice comprises: a capillary having defined therein a channel forreceiving said solution, and at least a structure placed inside of saidchannel, wherein at least said particular molecule which is chemicallyreactive with at least a substance in said solution is immobilized on aninterior surface of said channel, wherein said structure has a diameterwhich is in a range of 30 to 90% of a diameter of said channel.
 2. Thechemical reaction system according to claim 1, wherein, when a volumeallowed for a sample to flow in said channel is V1, an interior volumeof said channel is V2, and a ratio V1/V2 is in a range of 0.4 to 0.95.3. The chemical reaction system according to claim 1, wherein saidstructure is a fine particle or a strip.
 4. The chemical reaction systemaccording to claim 1, wherein said structure is a plurality of fineparticles arranged in said channel such that their centers are notaligned on one straight line.
 5. The chemical reaction system accordingto claim 1, wherein a flow of said solution in said channel is at leasta partly turbulent flow.
 6. The chemical reaction system according toclaim 1, wherein said particular molecule immobilized on the interiorsurface of said channel is a nucleic acid or a protein.
 7. The chemicalreaction system according to claim 1, wherein said channel is defined bya groove formed in a first substrate and a second substrate disposed tocover said groove.
 8. The chemical reaction system according to claim 1,wherein said channel is a lumen of a capillary.
 9. The chemical reactionsystem according to claim 1, wherein said particular molecule isimmobilized on at least an immobilization area in said channel, and adistance in a flow direction of said solution between a center of saidstructure and a center of said immobilization area is up to aboutthreefold a width of the channel in a plane substantially perpendicularto said solution flow direction.
 10. A chemical reaction method,comprising the steps of: preparing a reaction device having a particularmolecule immobilized on its interior surface, and having a structureaccommodated in its interior; introducing a solution into said reactiondevice; and allowing said particular molecule immobilized on theinterior surface to undergo a chemical reaction with a molecule includedin said solution, wherein in said step of chemical reaction, saidsolution moves in relation to said structure wherein said step ofpreparing the reaction device includes: providing a capillary havingdefined therein a channel for receiving said solution, providing atleast a structure placed inside of said channel, wherein said structurehas a diameter which is in a range of 30 to 90% of a diameter of saidchannel, and providing said particular molecule which is chemicallyreactive with at least a substance in said solution, wherein saidparticular molecule is immobilized on an interior surface of saidchannel.
 11. The chemical reaction method according to claim 10, whereina time required for said step of chemical reaction is at least about 10minutes.
 12. The chemical reaction method according to claim 10,wherein, when a volume allowed for a sample to flow in said channel isVI, an interior volume of said channel is V2, and a ratio V1/V2 is in arange of 0.4 to 0.95.
 13. The chemical reaction method according toclaim 10, wherein said structure is a fine particle or a strip.
 14. Thechemical reaction method according to claim 10, wherein said structureis a plurality of fine particles arranged in said channel such thattheir centers are not aligned on one straight line.
 15. The chemicalreaction method according to claim 10, wherein a flow of said solutionin said channel is at least a partly turbulent flow.
 16. The chemicalreaction method according to claim 10, wherein said particular moleculeimmobilized on the interior surface of said channel is a nucleic acid ora protein.
 17. The chemical reaction method according to claim 10,wherein said particular molecule is immobilized on at least animmobilization area in said channel, and a distance in a flow directionof said solution between a center of said structure and a center of saidimmobilization area is up to about threefold a width of the channel in aplane substantially perpendicular to said solution flow direction.